Sperm-disrupting activity of a root bark fraction of Flueggea virosa (Roxb. ex willd.) royle uncovered using LC-MS/MS profiling and functional evaluation

Introduction While Flueggea virosa (Roxb. Ex Willd.) Royle root bark is traditionally used in several parts of East Africa for fertility regulation, its contraceptive potential has not been systematically investigated. Given the paucity of safe, reversible, non-hormonal contraceptives for women, this study evaluated a root bark fraction for sperm-disrupting, intravaginal contraceptive activity using functional assays and a rabbit proof-of-concept model. Methods Following extraction and fractionation of authenticated root bark, the resulting extracts were evaluated for cytotoxicity in Vero cells and for disruption of human sperm functions by ascertaining immobilization, revival, viability, cervical mucus penetration and acrosin-related activity. Thereafter, chemical profiling of the most active fraction was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for descriptive annotation. Moreover, in vivo contraceptive efficacy and short-term local safety (10 days) were determined after intravaginal administration of the fraction in rabbits by histological examination of cervicovaginal tissues. Results The methanolic fraction (KBLM) demonstrated the strongest activity, completely immobilizing sperm, abolishing sperm revival after washing, markedly reducing sperm viability, strongly inhibiting cervical mucus penetration and suppressing acrosin-related activity at 1.62 mg/mL. In a rabbit proof-of-concept experiment, a single intravaginal dose prevented pregnancy at all tested concentrations (3.9, 7.8, and 15.6 mg/mL). Besides, repeated intravaginal exposure for 10 days revealed absent-to-mild cervicovaginal changes. Chemical fingerprinting putatively annotated flavonoid-, tannin-, and phenolic-related features, including catechin, quercetin derivatives, rutin, corilagin, and kaempferol glycosides, as prominent features of KBLM. Conclusion Our findings have demonstrated the sperm disruption activity of the methanolic fraction of F. virosa root bark. The observed disruption of multiple sperm functions, observed efficacy in a rabbit model and absent-to-mild short-term local tissue changes support additional preclinical investigations of the extract geared towards further functional characterization, extended safety testing, standardization, and potential formulation of an intravaginal contraceptive.